Bhat et al BMC Genomics (2020) 21:161 https://doi.org/10.1186/s12864-020-6574-4 RESEARCH ARTICLE Open Access Comparative milk proteome analysis of Kashmiri and Jersey cattle identifies differential expression of key proteins involved in immune system regulation and milk quality Shakil A Bhat1, Syed M Ahmad1* , Eveline M Ibeagha-Awemu2, Mohammad Mobashir3, Mashooq A Dar1, Peerzada T Mumtaz1, Riaz A Shah1, Tanveer A Dar4, Nadeem Shabir1, Hina F Bhat1 and Nazir A Ganai5 Abstract Background: Exploration of the bioactive components of bovine milk has gained global interest due to their potential applications in human nutrition and health promotion Despite advances in proteomics profiling, limited studies have been carried out to fully characterize the bovine milk proteome This study explored the milk proteome of Jersey and Kashmiri cattle at day 90 of lactation using high-resolution mass spectrometry based quantitative proteomics nanoscale LC-MS/Q-TOF technique Data are available via ProteomeXchange with identifier PXD017412 Results: Proteins from whey were fractionated by precipitation into high and low abundant proteins A total of 81 high-abundant and 99 low-abundant proteins were significantly differentially expressed between Kashmiri and Jersey cattle, clearly differentiating the two breeds at the proteome level Among the top differentiating proteins, the Kashmiri cattle milk proteome was characterised by increased concentrations of immune-related proteins (apelin, acid glycoprotein, CD14 antigen), neonatal developmental protein (probetacellulin), xenobiotic metabolising enzyme (flavin monooxygenase (FMO3), GLYCAM1 and HSP90AA1 (chaperone) while the Jersey milk proteome presented higher concentrations of enzyme modulators (SERPINA1, RAC1, serine peptidase inhibitor) and hydrolases (LTF, LPL, CYM, PNLIPRP2) Pathway analysis in Kashmiri cattle revealed enrichment of key pathways involved in the regulation of mammary gland development like Wnt signalling pathway, EGF receptor signalling pathway and FGF signalling pathway while a pathway (T-cell activation pathway) associated with immune system regulation was significantly enriched in Jersey cattle Most importantly, the high-abundant FMO3 enzyme with an observed 17-fold higher expression in Kashmiri cattle milk seems to be a characteristic feature of the breed The presence of this (FMO3) bioactive peptide/enzyme in Kashmiri cattle could be economically advantageous for milk products from Kashmiri cattle Conclusion: In conclusion, this is the first study to provide insights not only into the milk proteome differences between Kashmiri and Jersey cattle but also provides potential directions for application of specific milk proteins from Kashmiri cattle in special milk preparations like infant formula Keywords: Jersey, Kashmiri, Milk proteome, FMO3 enzyme * Correspondence: mudasirbio@gmail.com Division of Animal Biotechnology, Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-Kashmir, Srinagar, India Full list of author information is available at the end of the article © The Author(s) 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Bhat et al BMC Genomics (2020) 21:161 Background Bovine milk is a valued natural product which delivers a matrix of essential nutrients including growth and immune factors to offspring and a key raw material for human food preparations [1, 2] Some studies have characterized the bovine milk proteome, its bioactive profile, and the extent of cross reactivity of bovine bioactive milk peptides on various biological functions [3–7] Milk proteins are generally categorized into three major groups: caseins, whey proteins and milk fat globule membrane proteins [4, 8] Most of the polypeptides in milk are an essential source of amino acids to neonates [9] and many resist proteolysis [10, 11] Milk peptides also facilitate absorption of other nutrients in the gastro-intestinal tract, provide humoral immune responses and support intestinal development [12] Besides, digestion or fermentation of milk proteins also produces a number of bioactive peptides, which contribute as well to the various functional properties of milk [13, 14] The major proteins in milk are far outnumbered by numerous other minor proteins which play important roles in a wide range of physiological activities including antioxidant activity, postnatal development of new-borns, maturation of the immune system, establishment of symbiotic microflora, and protection against various pathogens [15, 16] Several studies have characterised the milk proteome in different species and breeds using different quantitative proteomic techniques [7, 16–20] The differences in the milk proteome profile have been attributed to genetic, management and disease factors [7, 21]) Although the diverse composition and biological functions of bovine milk has been reported extensively [22–24], the comparative abundance of milk proteins in Indian cattle breeds have not been investigated till date Kashmiri and Jersey cattle are two important milk animals which contribute significantly to the total milk production in the Indian northern state of Kashmir The Kashmiri cattle is an indigenous breed kept mainly for milk production in the hilly regions of Kashmir Kashmiri cattle are small, hardy and adapted to the hilly regions of Kashmir Whereas, Jersey is a well-established dairy breed imported to augment the milk production ability of Kashmiri cattle through cross breeding We hypothesize that the proteome profile of Kashmiri cattle milk may have special properties or differ from that of the well-established Jersey dairy breed due to its different genetic background and milk producing ability Therefore, the aim of this study was to study the protein profiles of Kashmiri and Jersey cattle milk which could reveal important protein factors underlying the physiological differences and differences in milk traits between the two breeds Results Proteome profile of bovine milk Proteins from whey were fractionated by precipitation into high and low abundant proteins A total of 180 Page of 10 proteins were differentially expressed (DE) (FDR < 0.1) between Kashmiri and Jersey cattle Specifically, 91 and 89 proteins were significantly upregulated (FDR < 0.1) in Kashmiri and Jersey cattle, respectively (Additional file 2: Table S2a and S2b, Additional file 3) The most upregulated high abundant proteins (fold change (FC) > 2) were CSN2, CD4 and LF, and low abundant proteins were FMO3, GLYCAM1, APLN and BTC in Kashmiri cattle (Table 1, Fig 1) Whereas, LALBA, ZNF496, CSN3 and LGB were the most upregulated high abundant proteins and RAC1, B2M and SAR1B were the most upregulated minor milk proteins in Jersey cattle (Table 1) Enriched gene ontology terms of significantly upregulated proteins in Kashmiri and Jersey cattle Gene ontology (GO) enrichment of significantly upregulated proteins in Kashmiri and Jersey cattle found a total of enriched GO terms in Kashmiri and in Jersey cattle (Table 2) Only extracellular region (GO:0005576) reached significance after FDR correction in both breeds (Table 2) Protein categories identified through GO annotation The identified differentially upregulated proteins in Kashmiri and Jersey cattle were categorized according to their GO annotation (Additional file 2: Table S103) Most of the significantly upregulated proteins in both cattle breeds were enzyme modulators (SERPINA3, BTN1A1, SERPINC1, SERPINF2, Serin peptidase inhibitor, RAC1, RRAS, BTN 1A1 and uterine milk protein) and hydrolases (GNB2, CT SD, GNB1, PNLIPRP2, CYM) (Fig a and b) However, proteins belonging to the chaperone classes (HSP90AA1, YWHAB, YWHAZ) were significantly upregulated in Kashmiri cattle only (Fig 2a and b) Enriched pathways by significantly upregulated proteins in Kashmiri and Jersey cattle Significantly upregulated proteins in Kashmiri and Jersey cattle were enriched to 12 and pathways at uncorrected P < 0.05, respectively (Table 3) When FDR correction was applied, 10 and one proteins remained significant (FDR < 0.1) in Kashmiri and Jersey cattle, respectively (Table 3) Of all the pathways, only EGF receptor signalling pathway was enriched at uncorrected P < 0.05 by significantly upregulated proteins in both breeds Discussion The present study was designed to characterize and compare the milk proteome of Kashmiri and Jersey cattle Over the past few decades, interest to reveal the dynamics of milk proteome has grown and there have been remarkable developments in the techniques used for fractionation and identification of proteins [25–27] In the present study, a combination of fractionation and mass spectrometry Bhat et al BMC Genomics (2020) 21:161 Page of 10 Table Significantly upregulated high abundant and low abundant milk proteins in Kashmiri and Jersey cattle Accession No Kashmiri Cattle Protein Gene ID FC P-value FDR Significantly upregulated abundant milk proteins J9UHS4 Beta-casein CSN2 2.74 0.044 0.055 Q8HY42 CD4 antigen CD4 2.09 0.039 0.043 E6YCQ7 Lactoferrin LF 2.04 0.037 0.047 Significantly upregulated less abundant milk proteins Jersey cattle Q8HYK4 Flavin-containing monooxygenase FMO3 16.6 0.041 0.050 P80195 Glycosylation-dependent cell adhesion molecule GLYCAM1 7.93 0.037 0.047 P30932 CD9 antigen CD9 7.24 0.038 0.048 Q9TUI9 Apelin APLN 3.63 0.046 0.050 Q9TTC5 Probetacellulin BTC 2.97 0.037 0.042 Q9TRC0 Enterotoxin-binding glycoprotein PP16K N/A 2.91 0.038 0.048 Q3SZR3 Alpha-1-acid glycoprotein ORM1 2.66 0.046 0.050 C4PU73 Serin peptidase inhibitor, clade A LOC286871 2.53 0.039 0.046 Q9TS52 Adipocyte differentiation-related protein N/A 2.53 0.042 0.055 P46201 Uterine milk protein N/A 2.41 0.043 0.049 Q5GN72 Alpha-1-acid glycoprotein AGP 2.07 0.037 0.040 Beta-lactoglobulin LGB 7.24 0.037 0.051 A0A140T8A9 Kappa-casein CSN3 4.17 0.04 0.046 F6I8C5 Zinc finger protein 496 ZNF496 2.33 0.037 0.061 G9G9X6 Alpha-lactalbumin LALBA 2.11 0.038 0.041 Significantly upregulated abundant milk proteins P02754 Significantly upregulated less abundant milk proteins G8FZ88 ATP synthase subunit A N/A 4.09 0.037 0.047 P62998 Ras-related C3 botulinum toxin substrate RAC1 3.85 0.044 0.067 P01888 Beta-2-microglobulin B2M 2.85 0.039 0.041 Q3T0T7 GTP-binding protein SAR1b SAR1B 2.2 0.037 0.046 Q9XSC9 Transcobalamin-2 TCN2 2.18 0.044 0.051 Q95114 Lactadherin MFGE8 2.11 0.039 0.049 techniques were used to comprehensively characterize the milk proteome profiles of Kashmiri and Jersey cattle breeds A total of 180 proteins were found to be differentially expressed between Kashmiri and Jersey cattle Interestingly, 90 and 89 of the differentially expressed proteins were significantly upregulated in Kashmiri and Jersey cattle, respectively Enzyme modulators were the major class of up-regulated proteins in both Kashmiri (20.51%) and Jersey cattle (14.28%) Hydrolases represented 12.82 and 14.28% of upregulated proteins in Kashmiri and Jersey cattle, respectively Interestingly, chaperone class of proteins was only observed in milk of Kashmiri cattle Chaperones help in the folding of newly synthesized proteins and prevent their premature (mis) folding at least until a domain capable of forming a stable structure is synthesized As expected and in agreement with earlier studies ([26, 27]), the casein and whey fraction proteins were highly expressed in both breeds However, a different set of high abundant milk proteins were significantly upregulated in each of the breeds For example, the abundantly expressed proteins beta-casein, lactoferrin and CD4 were significantly upregulated in Kashmiri while beta-lacto globulin, kappacasein and alpha-lactalbumin were significantly upregulated in Jersey (Table 1) Interestingly, the low abundant proteins FMO3, GLYCAM1, CD9, APLN, BTC, enterotoxinbinding glycoprotein PP16K, ORM1, serin peptidase inhibitor clade A, adipocyte differentiation-related protein and uterine milk protein were significantly upregulated in Kashmiri while ATP synthase subunit A, RAC1, B2M, SAR1B, TCN2 and MFGE8 were upregulated in Jersey These Bhat et al BMC Genomics (2020) 21:161 Page of 10 Fig Volcano plot of differentially expressed proteins between Kashmiri and Jersey cattle Red points indicate more abundant proteins in Kashmiri cattle; blue points indicate more abundant proteins in Jersey cattle Table Gene ontology terms enriched for significantly upregulated proteins in Kashmiri and Jersey cattle Functions Description GO term No of proteins Protein IDs Gene IDs P-value FDR Kashmiri Cattle Catalytic activity GO:0003824 18 P19120, P12763, P30122, P11017, Q8MK44, P80209, P62871, Q8HXQ5, Q4GZT4, Q0IIG8, A5PK46, P00794, P80929, Q0VCZ8, Q2UVX4, P80025 HSPA8, AHSG, CEL, GNB2, DGAT1, CTSD, GNB1, ABCC1, ABCG2, RAB18, PNLIPRP2, CYM, ANG2, ACSL1, C3, LPO 0.0002 0.44 Antioxidant activity GO:0016209 P80025 LPO 0.0843 0.79 Membrane GO:0016020 P19120, P30122, Q8MK44, P80209, Q8HXQ5, Q4GZT4, Q0IIG8, P00794, P02702, P30932, P18892 HSPA8, CEL, DGAT1, CTSD, ABCC1, ABCG2, RAB18,CYM, FOLR1,CD9,BTN1A1 0.0198 0.181 Extracellular region GO:0005576 10 P46201, P02666, P30122, C4PU73, Q9TTE1, P21214, P80025 Uterine milk protein, CSN2, CEL, Serin peptidase inhibitor, SERPINA3–1,TGFB2,LPO 0.00111 0.0354 Reproduction GO:0000003 A0A140T8A9, P11151, P02668, A5PK46 CSN3, LPL, CSN3, 0.005 Catalytic activity GO:0003824 22 Q8HYJ9, Q5E9R3, P11151, Q5E9B1, Q8MK44, P62998, Q2TBH2, Q8HXQ5, Q148J4, F1MN60, P101, Q4GZT4, A5PK46, P00794, P80457, P02754, P80025 FMO3, EHD1, LPL, LDHB, DGAT1, 0.066 RAC1, RRAS, ABCC1, RAB2A, ATP2B2, ANG1, ABCG2, PNLIPRP2, CYM, XDH, LGB, LPO 0.83 Antioxidant activity GO:0016209 P80025 LPO 0.087 0.52 Extracellular region GO:0005576 11 P17697, A0A140T8A9, P34955, P46201, P02663, Q3ZCH5, P41361, P28800, C4PU73, P02662,P02668,P21214,P80025 CLU, CSN3, SERPINA1, Uterine milk protein, CSN1S2, AZGP1, SERPINC1, SERPINF2, Serin peptidase inhibitor, CSN1S1, CSN3, TGFB2, LPO 0 Molecular Cellular Jersey Cattle Molecular Cellular 0.422 Bhat et al BMC Genomics (2020) 21:161 Page of 10 Fig Classification of differentially expressed proteins in Kashmiri and Jersey cattle by gene ontology annotation (a) Protein classes (upregulated proteins only) in Kashmiri cattle and (b) Jersey cattle results indicate a clear distinction as well as wide differences in the proteome profiles between the breeds which could be explained by high selection pressure for milk production traits in Jersey The differences in the expression of high abundant proteins between the breeds might confer differential benefits to their milks For example, different levels of phosphorylation of beta-casein has been reported to affect the availability of calcium and protein micelle stability of milk [28], which might have important consequences on the nutrition and technological properties of milk and milk products Additionally, other key bioactive proteins identified in this study that are well known to exert beneficial effects on human nutrition and health include lactoferrin, GLYCAM1, betacellulin, apelin, LALBA and serine peptidase inhibitor, etc Iron sequestering properties of lactoferrin (LF), along with blockade of microbial carbohydrate metabolism and destabilisation of the bacterial cell wall [29, 30], has been shown to produce bactericidal and bacteriostatic effects in a wide range of microorganisms, including gram positive and gram negative bacteria, aerobes, anaerobes, yeasts and parasites [31–33] Similarly, GLYCAM1 with a 7.93-fold expression in Kashmiri cattle is known to act as an antimicrobial peptide with ability to protect the intestinal mucosal tract of neonates largely due to its lubricating properties [34, 35] In addition to these, apelin peptides might be involved in maturation of the gastrointestinal tract [36, 37] Betacellulin (BTC), a key epidermal growth factor (EGF) [38] might regulate the development and maturation of the neonatal gut and immune system [39] EGFs are major growth promoting factors in human milk [40] but the biological significance of BTC in bovine milk is currently unclear and needs further investigation However, one plausible explanation for the presence of BTC in bovine milk might be to stimulate the proliferation of the gastrointestinal epithelia in new-borns, as has been proposed for milk-borne EGF and TGF-α (Transforming growth factor alpha) in other species [41] With respect to Jersey breed, peptides resulting from partial digestion of high abundant proteins such as LALBA, CSN2 and CSN3 in the small intestine may influence gut functions including immune stimulation, mineral and trace element absorption and host defence against infection [42] Alpha-lactalbumin enhances infant gastrointestinal function [43], motility and antimicrobial activity [44] CSN3 is readily hydrolysed in calf’s stomach, allowing the formation of a coagulum that can be readily digested [45] and also provides heat stability to milk by stabilising the casein micelle [45] Moreover, CSN3 prevents infection by disrupting the attachment of pathogens to mucosal cells [46] CSN3 digestion results in the formation of a glycomacropeptide which in turn enhances mineral absorption [47] Bovine beta 2-microglobulin (B2M) is an antibacterial protein present in milk fat globules B2M possesses potent antibacterial activities against Gram positive pathogenic bacteria [48] Bovine milk is an abundant source of bioavailable B12 vitamin wherein when complexed with transcobalamin, a major vitamin B12 binding protein in cows’ milk [49], stimulates vitamin B12 absorption through intestinal epithelial cells [50] Lactadherin is secreted by mammary epithelial cells and stored in milk fat globules [51] Lactadherin, as one of the immune components in bovine milk has been found to prevent rota viral infection in infants by removing the sialic acid from the viral coat [52, 53] It is worthwhile to note that the low abundant protein, flavin-containing monooxygenase (FMO3) had 16.6 fold expression rate in Kashmiri as compared to Jersey This is the first report wherein FMO3 has been found to be highly expressed in Kashmiri cattle Increased presence of FMO3 might be important due to its ability to oxidise trimethylamine (TMA), a compound with fishy odour, to TMAO (Trimethylamine N-oxide), an odourless oxide Absence of FMO3 leads to fishy flavour in milk due to increased buildup of TMA, and thus might play an important role in maintaining the quality of milk [54–56] Moreover, FMO3 Bhat et al BMC Genomics (2020) 21:161 Page of 10 Table Enriched pathways by upregulated proteins in Kashmiri and Jersey cattle Pathway Proteins p-value FDR Proteins Genes 0.1719 P11017, P62871 GNB2, GNB1 Kashmiri cattle Beta3 adrenergic receptor signaling pathway (P04379) 0.07265 Beta2 adrenergic receptor signaling pathway (P04378) 0.09576 0.8552 P11017, P62871 GNB2, GNB1 Metabotropic glutamate receptor group III pathway (P00039) 0.0124 0.0723 P11017, P62871 GNB2, GNB1 Beta1 adrenergic receptor signaling pathway (P04377) 0.00599 0.0543 P11017, P62871 GNB2, GNB1 5HT4 type receptor mediated signaling pathway (P04376) 0.07387 0.1987 P11017, P62871 GNB2, GNB1 5HT2 type receptor mediated signaling pathway (P04374) 0.0141 0.0792 P11017, P62871 GNB2, GNB1 5HT1 type receptor mediated signaling pathway (P04373) 0.09671 0.1897 0.049 P11017, P62871 GNB2, GNB1 Integrin signalling pathway (P00034) 0.0823 0.353 P63258, E1BBG2 ACTG1, MICALL1 Heterotrimeric G-protein signaling pathway-Gi alpha and Gs alpha mediated pathway (P00026) 0.0658 0.298 P11017, P62871 GNB2, GNB1 Wnt signaling pathway (P00057) 0.0388 0.197 P63258, P11017, P62871 GNB2, GNB1, ACTG1 Thyrotropin-releasing hormone receptor signaling pathway (P04394) 0.082 0.759 P11017, P62871 GNB2, GNB1 FGF signaling pathway (P00021) 0.0417 0.2 P63103, P68250 YWHAZ,YWHAB EGF receptor signaling pathway (P00018) 0.041 P63103, Q95115, P68250, Q9TTC5 YWHAZ, STAT5A, YWHAB, BTC PI3 kinase pathway (P00048) 0.002 0.0685 P63103, P11017, P62871 GNB2, GNB1, YWHAZ Opioid prodynorphin pathway (P05916) 0.00406 0.0602 P11017, P62871 GNB2, GNB1 Histamine H1 receptor mediated signaling pathway (P04385) 0.00647 0.0502 P11017, P62871 GNB2, GNB1 Enkephalin release (P05913) 0.00387 0.0701 P11017, P62871 GNB2, GNB1 Angiotensin II-stimulated signaling through G proteins and beta-arrestin (P05911) 0.00488 0.0497 P11017, P62871 GNB2, GNB1 CCKR signaling map (P06959) 0.0816 0.36 GNB1,YWHAB Metabotropic glutamate receptor group II pathway (P00040) 0.00647 0.0527 P11017, P62871 GNB2, GNB1 0.0879 0.796 P62998, Q2TBH2 RAC1, RRAS P62871, P68250 Jersey Cattle Integrin signalling pathway (P00034) EGF receptor signaling pathway (P00018) 0.00663 0.36 P62998, Q2TBH2, Q95115 RAC1, RRAS, STAT5A PDGF signaling pathway (P00047) 0.0567 0.66 Q148J4, Q95115 RAB2A, STAT5A Blood coagulation (P00011) 0.000428 0.0698 P34955, P41361, P28800 SERPINA1, SERPINC1, SERPINF2 CCKR signaling map (P06959) 0.0872 CLU, RAC1 0.836 P17697, P62998 T cell activation (P00053) 0.0318 0.648 P01888, P62998 B2M, RAC1 TGF-beta signaling pathway (P00052) 0.0298 0.694 Q2TBH2, P21214 RRAS, TGFB2 belongs to a drug metabolising enzyme class with ability to oxidize xenobiotics, pesticides and other foreign inhabitants in body fluids including milk and serum [57–60] and hence presents an efficient defence mechanism in newborns The presence of FMO3 at high concentrations in Kashmiri cattle milk can favour utilization of Kashmiri cattle milk in commercial preparations for the promotion of human health and nutritional status In fact, bio-mining of such bioactive milk protein constituent and marketing it as ingredients may not only serve as a lucrative business for the Indian dairy industry but also in the development of products for consumers with special needs like allergy and milk tolerance The GO analysis of significantly up-regulated proteins revealed only one significantly enriched GO term (extracellular region) after FDR correction in both breeds and limited functional overlap was found between the present proteomic data and our earlier transcriptome data [61] Bhat et al BMC Genomics (2020) 21:161 indicating the failure of RNA-based analyses to represent completely protein dynamics [62] Pathway analysis helps in biological interpretation of proteomic and other high-throughput data in cells or organisms [63] Most of the pathways (Wnt signaling pathway, EGF receptor signaling pathway, FGF signaling pathway, PI3 kinase pathway) significantly enriched by the significantly upregulated proteins in Kashmiri cattle are involved in mammary gland development Wnt signaling pathway regulates mammary development [64] during various stages of mammary morphogenesis [65] The proteins enriched in the Wnt signalling pathway were GNB1(G protein subunit beta 1), GNB2 (G protein subunit bBeta 2) and ACTG1(actin gamma 1) ACTG1 plays a critical role in branching and alveolar development of the mammary gland through cytoskeletal remodelling [66] FGF signalling pathway controls mammary epithelial cell branching and morphogenesis [67] and activates PI3 kinase pathway through phosphorylation [68] Epidermal growth factor family plays essential roles in regulating cell proliferation, survival and differentiation of mammary epithelial cells through STAT5A, a key non-tyrosine kinase protein indirectly regulated by JAK2/ELF5, insulin growth factor, estrogen, and progesterone signalling pathways [69] In Jersey cattle, two significantly (p < 0.05) enriched pathways, blood coagulation/coagulation cascades and T cell activation pathways are associated with immune system regulation [70] SERPINA1, SERPINC1, SERPINF2 are important proteins in blood coagulation pathway whereas, B2M and RAC1 play critical roles in T cell activation pathway These proteins play fundamental roles in innate immunity in addition to enhancing adaptive immune responses [71] Altogether, a wide range of proteins were detected in this study including proteins involved in immune response, host defense and milk quality as well as qualitative and quantitative differences in their milk proteome Conclusion A total of 91 and 89 proteins were significantly upregulated in Kashmiri and Jersey cattle, respectively A different set of high- abundant and low-abundant proteins were significantly upregulated in Kashmiri and Jersey cattle, clearly differentiating the two breeds at the proteome level Immune-related proteins (CD4, LF and GLYCAM 1) and drug metabolising enzyme (FMO3) were abundantly expressed in Kashmiri cattle milk The presence of FMO3 at high concentrations in Kashmiri cattle milk could favour its utilization in commercial preparations for human health promotion and consequently serve as a boost for increased business opportunities for the Indian dairy industry Page of 10 Methods Experimental animals and sampling The ethical clearance was approved by the Institutional Animal Ethics Committee (IAEC) of Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir A total of three healthy Kashmiri and three Jersey cows in their 3rd lactation from the university dairy farm (Mountain Livestock Research Institute, Share-Kashmir University of Agricultural Sciences and Technology of Kashmir, India) were selected for the study The animals were kept under similar feeding and management conditions to minimise environmental variation Fresh milk samples (200 mL) were aseptically collected from all the four quarters (50 mL per quarter) at day 90 in milk (D90), mixed thoroughly, placed on ice and immediately transported to the laboratory for further analysis Protein preparation Milk samples were processed differently for high and low abundance protein analysis For high-abundance protein analysis, 50 mL of milk was immediately placed on ice after collection followed by centrifugation at 4000×g for 10 at °C within h of collection The fat layer was removed and skimmed fraction was stored at − 20 °C Whereas, for low abundance protein analysis, 0.24 mL (100X) mammalian protease inhibitor cocktail (Sigma, Milwaukee, WI, USA) was added to 50 mL of milk followed by centrifugation at 4000×g for 15 at °C The cream layer was removed and the skimmed or whey portion was depleted of casein using a previously described method [72] Briefly, 60 mM CaCl2 was added to skimmed sample and the pH was adjusted to 4.3 using 30% acetic acid (Fisher Scientific, Fair Lawn, NJ, USA) Samples were then centrifuged at 189, 000×g at °C for 70 and the supernatant was collected and stored at − 80 °C Enrichment of low abundance proteins Low abundance minor proteins were enriched using the ProteoMiner Kit (BioRad Laboratories, Hercules, CA, USA) as per manufacturer’s protocol Whey samples were placed in individual ProteoMiner columns, mixed thoroughly by shaking (gently) followed by incubation at room temperature for h Subsequently, samples were washed thoroughly using HPLC grade water to remove excess proteins by centrifugation at 7000 g for Low abundance proteins were eluted off the beads by addition of 20 μl x Laemmli sample buffer (8% SDS, 40% glycerol, 250 mM Tris, pH 6.8, 400 mM DTT with trace amount of bromophenol blue) In-solution digestion of proteins and nano-scale LC/MS analysis on QTOF The pellets after acetone precipitation (high abundant proteins) or TCA (Trichloroacetic acid)-acetone precipitation ... the milk proteome profiles of Kashmiri and Jersey cattle breeds A total of 180 proteins were found to be differentially expressed between Kashmiri and Jersey cattle Interestingly, 90 and 89 of. .. abundant proteins and RAC1, B2M and SAR1B were the most upregulated minor milk proteins in Jersey cattle (Table 1) Enriched gene ontology terms of significantly upregulated proteins in Kashmiri and Jersey. .. 21:161 Page of 10 Fig Volcano plot of differentially expressed proteins between Kashmiri and Jersey cattle Red points indicate more abundant proteins in Kashmiri cattle; blue points indicate more